Giovanni Grasso

SOMATOSTATIN AND VASOACTIVE INTESTINAL PEPTIDE REDUCE INTERFERON GAMMA PRODUCTION BY HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS

There is increasing evidence that neuropeptide modulation of the immune response is an important physiological phenomenon which involves the interaction of peptidergic neuromodulators with specific neuropeptide receptors on the plasma membrane of immune effector cells. Many studies have examined the effect of neuropeptides on mitogen-induced lymphocyte proliferation and immunoglobulin synthesis but very little is known about specific lymphokine production. In this study, we describe the effect of somatostatin (SOM) and vasoactive intestinal peptide (VIP) on interferon gamma (IFN-gamma) production by normal human peripheral blood mononuclear cells (PBMC) stimulated in vitro with polyclonal T cell activator staphylococcal enterotoxin A (SEA). Our findings provide experimental evidence that both SOM and VIP reduce the IFN-gamma production by SEA-stimulated PBMC. This reduction was time- (with maximal effect at 72 h) and dose-dependent (at doses as low as 10(-11) M with maximal effect at concentrations between 10(-9) and 10(-8) M of neuropeptides). This effect was absent in resting PBMC. The meaning of inhibitory effect of VIP and SOM on IFN-gamma production and its role in immune response in vivo are discussed.

Glucocorticoid receptors in human peripheral blood mononuclear cells in relation to age and to sport activity.

Glucocorticoid receptors (GR) are ubiquitous molecules and are present also in the hippocampus and in several other nervous and immune tissues. Peripheral blood mononuclear cells (PBMCs) are a good model for studies of GR in humans. Glucocorticoids are important for maintaining cellular and humoral homeostasis and are key mediators of neuroendocrine-immune regulatory interactions. The increase of cortisol is immunosuppressive and reduces GR concentration both in nervous and immune systems. Variation of glucocorticoids in healthy aged subjects and athletes has been shown. Prompted by these results, we have investigated in man a possible relationship between GR binding capacity in the PBMCs and age, in relation also to plasma testosterone and cortisol. The same parameters have been examined in a group of soccer players for comparison with the sedentary group. GR binding capacity was higher in younger subjects than in older ones, and lower in the group of athletes than in the younger and older sedentary subjects. In the sedentary group a negative correlation was present between GR binding capacity and age. Plasma cortisol was higher and testosterone lower in the athletes; they were negatively correlated in athletes and positively correlated in the sedentary subjects. The results for athletes agree with their lower anabolic/catabolic balance. The mechanism of reduced GR levels in relation to age and sport activity could involve a loss or an involution of receptor synthesis. However other possibilities, such as altered distribution of lymphocyte subpopulations with different receptor concentrations and with different cytokine production, cannot be excluded. Several neuroendocrine-immune interactions could be responsible for reduced GR levels with age and sport activity in man.

The effect of LHRH and TRH on human interferon-gamma production in vivo and in vitro.

Accumulating evidence suggests that hypothalamic luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH) are two hypophysiotropic factors which modulate the immune response. The aim of the present study was to determine the in vivo effects of an intravenous bolus of LHRH and TRH on plasma interferon (IFN)-gamma production in five normoprolactinemic women with irregular menstrual cycles. We also determined prolactin (PRL), thyrotropin (TSH), follicle stimulating hormone (FSH), and luteinizing hormone (LH) levels before and after intravenous administration of LHRH and TRH. The results demonstrate that intravenous bolus of LHRH/TRH increases plasma IFN-gamma levels, with the maximum response 45 min after in vivo administration of hypothalamic peptides and after peak levels of adenohypophyseal hormones (PRL: 15 min; TSH: 30 min; FSH: 30 min; LH: 30 min). In order to investigate a possible direct action of hypothalamic hormones on immune cells, we also evaluated, in the same subjects, the influence of LHRH and TRH on IFN-gamma production by human peripheral blood mononuclear cells (PBMCs), collected before the intravenous administration of the peptides and stimulated in vitro with bacterial superantigen staphylococcal enterotoxin A (SEA) and concanavalin A (Con A). LHRH and TRH, separately and together, significantly enhanced in vitro IFN-gamma production by SEA- and ConA-activated PBMCs. The present results suggest that hypothalamic peptides (LHRH and TRH) directly, and/or indirectly pituitary hormones (PRL, TSH, FSH, and LH) or IL-2, have stimulatory effect on IFN-gamma producing cells and are further evidence of interactions between the neuroendocrine and immune systems.

The lymphatic route. 1) Albumin and hyaluronidase modify the normal distribution of interferon in lymph and plasma.

When human recombinant interferon-α2 diluted in saline was injected s.c. into rabbits, the total amount recovered in thoracic lymph was less than 0.4%. Recoveries increased from 2- to 8-fold if interferon was injected in 4% albumin or with hyaluronidase, respectively. Albumin added to interferon acts as an interstitial fluid expander, thus favoring interferon absorption through lymphatics rather than blood capillaries. This strategy may increase the therapeutic index of interferon.

Estrogen and μ-opioid receptor antagonists counteract the 17β-estradiol-induced licking increase and interferon-γ reduction occurring during the formalin test in male rats

&NA; Women have a higher incidence of chronic pain syndromes than men and are generally more sensitive to experimental pain. Numerous studies have shown that the female gonadal hormones, estrogens, can profoundly affect the nervous and immune systems, including mechanisms involved in pain and nociception. In the present study, we used antagonists of estrogen receptors (ER) or &mgr;‐opioid receptors (&mgr;OR) to evaluate the effects of estrogens on formalin‐induced behavioural and immune responses in male rats. After two days of priming with 17&bgr;‐estradiol or saline (i.c.v.), animals were subjected to the formalin test; 15 min prior to formalin (50 &mgr;l, 5%) or sham injection in the hind paw, animals were treated with an ER antagonist (ICI 182,780, ICI) or a &mgr;OR antagonist (&bgr;‐funaltrexamine, FNA) or saline. The spontaneous behaviours, pain‐related behaviours and interferon‐&ggr; (IFN‐&ggr;) production by peripheral blood mononuclear cells were studied in all groups. We found that central administration of estradiol increased the amount of licking of the formalin‐injected paw in the second phase of the formalin test. Whereas ICI and FNA had no effect on pain behaviour in saline‐pre‐treated animals, both antagonists reversed the estradiol‐induced increase in licking. The immune system was differently affected by formalin and estradiol treatment. Indeed, formalin injection per se decreased IFN‐&ggr; production; estradiol had no effect on sham‐injected animals but strongly reduce the decrease of IFN‐&ggr; production in formalin‐injected animals. The results demonstrate that centrally acting estrogens affect ER‐ and &mgr;OR‐mediated pain processing and influence immune function.

Effects of Lactobacilli on Interferon Production in Young and Aged Micea

It is well established that yogurt bacteria have immunomodulatory and immunostimulating activities in experimental animals and man. In mice, a diet supplemented with yogurt containing viable lactobacilli induces an increase in antibody production, a modification of splenocyte surface antigens, and an increase in their proliferative response to PHA and ConA.I.* Furthermore, it potentiates the hosts cell-mediated immune response by increasing the percentage of B lymphocytes and the PHAand LPS-induced proliferative responses of Peyers patch cell suspension^.^ In vitro lactobacilli increase interferon-y (IFN-y) production by ConA-stimulated human peripheral blood mononuclear cells and NK activity! The mechanism responsible for these immunomodulatory properties of lactobacilli is not clear, nor are their effects on IFN production in vivo. Because reduction of cytokine production with age, primarily IFN-y,S*6 IFN-P,6 interleukin-1 (IL-l),7-9 and IL-2,1°Jl has been well documented, in the present study, we evaluated the effect on IFN production of supplementing the diet of young and aged mice with live Lactobacillus bulgaricus and Streptococcus thermophilus.

Differential expression of follistatin and FLRG in human breast proliferative disorders

BackgroundActivins are growth factors acting on cell growth and differentiation. Activins are expressed in high grade breast tumors and they display an antiproliferative effect inducing G0/G1 cell cycle arrest in breast cancer cell lines. Follistatin and follistatin- related gene (FLRG) bind and neutralize activins. In order to establish if these activin binding proteins are involved in breast tumor progression, the present study evaluated follistatin and FLRG pattern of mRNA and protein expression in normal human breast tissue and in different breast proliferative diseases.MethodsParaffin embedded specimens of normal breast (NB - n = 8); florid hyperplasia without atypia (FH - n = 17); fibroadenoma (FIB - n = 17); ductal carcinoma in situ (DCIS - n = 10) and infiltrating ductal carcinoma (IDC - n = 15) were processed for follistatin and FLRG immunohistochemistry and in situ hybridization. The area and intensity of chromogen epithelial and stromal staining were analyzed semi-quantitatively.ResultsFollistatin and FLRG were expressed both in normal tissue and in all the breast diseases investigated. Follistatin staining was detected in the epithelial cytoplasm and nucleus in normal, benign and malignant breast tissue, with a stronger staining intensity in the peri-alveolar stromal cells of FIB at both mRNA and protein levels. Conversely, FLRG area and intensity of mRNA and protein staining were higher both in the cytoplasm and in the nucleus of IDC epithelial cells when compared to NB, while no significant changes in the stromal intensity were observed in all the proliferative diseases analyzed.ConclusionThe present findings suggest a role for follistatin in breast benign disease, particularly in FIB, where its expression was increased in stromal cells. The up regulation of FLRG in IDC suggests a role for this protein in the progression of breast malignancy. As activin displays an anti-proliferative effect in human mammary cells, the present findings indicate that an increased FST and FLRG expression in breast proliferative diseases might counteract the anti-proliferative effects of activin in human breast cancer.

Immune and neuroendocrine response to restraint in male and female rats

A parallel study of the modification in the opioid and immunological systems induced by acute restraint (RT) was carried out in male and female rats 24 hr after the treatment. beta-Endorphin-like immunoreactivity (beta-ELI) was measured in two brain areas (ventral hypothalamus [HYP] and periaqueductal gray matter [PAG]) and in the pituitary (anterior and neurointermediate lobes), together with plasma corticosterone (C) and ACTH. Immune function was measured as mitogen-induced Interferon-gamma (IFN-gamma) production by splenocytes. RT reduced beta-ELI levels in the PAG in males and females. Plasma levels of C and ACTH did not differ from the basal levels in restrained animals. RT reduced IFN-gamma production in both sexes, but this effect was more marked in females than in males. The possible relationship between the immune and opioid system is discussed.

Endothelin receptor A expression in human inflammatory cells

Most inflammatory diseases show elevated levels of endothelin-1 (ET-1) probably due to an alteration in vascular structure and function with activation/accumulation of inflammatory cells. The ET receptors (ET(A), ET(B)) are widely expressed in all human vessels, consistent with the main role of ET-1 in maintaining physiological vascular tone. Previous findings have shown the expression on inflammatory cells such as neutrophils (PMNs) and macrophages (MØs) of ET-1 and endothelin-converting enzyme-1 (ECE-1) (the key enzyme in the biosynthesis of ET-1). Therefore the role of ET-1 cannot be related only to the vasoactivity. Our study was aimed to determine the expression and the cellular location of ET receptors in both human PMNs and MØs by the use of RT-PCR assay, Western blot analysis and immunocytological methods. Our results showed for the first time that PMNs and MØs clearly expressed ET(A) (mRNA and protein). Considering that the overproduction of ET-1 following endothelial dysfunction and inflammation, contributes to pathophysiological processes such as vascular hypertrophy, cell proliferation and fibrosis, our results suggest that PMNs and MØs can also play a key role in vascular dysfunctions via the possible formation of an autocrine loop between ET-1 and ET(A).

Glucocorticoid receptor mRNA expression in peripheral blood mononuclear cells in high trained compared to low trained athletes and untrained subjects

Background: Physiological needs during prolonged exercise are a potent stimulus for the hypothalamic-pituitaryadrenal (HPA) axis. Hence, athletes undergoing daily endurance training sessions may have frequent and prolonged phases of endogenous hypercortisolism. Since chronic glucocorticoids treatment leads to down-regulation of glucocorticoid receptor α (GR-α) mRNA expression, endurance training could lead to modulation of GR expression. Aim: The aim of the study was to evaluate GR-α and GR-β mRNA expressions in peripheral blood mononuclear cells and plasma cortisol, ACTH and cortisol binding globulin (CBG) concentrations at rest in subjects undergoing different training regimes. Subjects and methods: Nine high trained (HT) swimmers (training volume: 21.6±1.7 hours/week in 10–12 sessions) were compared with two age-matched control groups represented by 8 low trained (LT) runners (training volume: 6.4±2.6 h/week in 3–5 sessions) and 9 untrained subjects. Expression of GR was determined by RT-PCR of total RNA. Hormone levels were determined by radioimmunoassay methods. Results: HT athletes showed 10 times less GR-a mRNA expression than the untrained subjects, while LT athletes exhibited values about twofold less than the untrained subjects. GR-β mRNA expression was undetectable in all subjects. No differences were observed among the three groups in hormone levels. Conclusions: GR-a mRNA expression is repressed in proportion to the amount and frequency of the stressful stimuli due to training. Hence, this down-regulation may be a consequence of the frequent and prolonged exposure to cortisol acute elevations induced by training. GR-β did not play an important role in inducing the down-regulation of GR-a mRNA expression observed.